Essential heavy metals, such as copper and zinc, are required as cofactors in redox reactions and ligand interactions and they also participate in charge stabilization, water ionization, and charge shielding during biocatalysis (Voet and Voet, 1995, In: Biochemistry, 2nd ed., John Wiley and Sons, Inc., New York). In addition to the essential heavy metals, non-essential heavy metals, such as arsenic, cadmium, mercury and lead,.are found in natural mineral deposits or as a result of human activity and they are frequently encountered by living organisms (Nriagu and Pacyna, 1988, Nature 333:134-138). Both essential and non-essential heavy metals can pose an acute problem for all living organisms in that the organisms often encounter supraoptimal concentrations of essential heavy metals and excess micromolar concentrations of non-essential heavy metals such as As, Cd, Hg and Pb. High concentrations of these non-essential heavy metals exert toxic effects through the displacement of endogenous metal cofactors, heavy or otherwise, from their cellular binding sites, aberrant reactions with the thiol groups of proteins and coenzymes, and the promotion of the formation of active oxygen species (AOS; Stadtman, 1990, Free Radic. Biol. Med. 9:315-325).
Massive global expansion in industrial and mining activities during the last two decades combined with changes in agricultural practices, have markedly increased contamination of groundwaters and soils with heavy metals. Indeed, it is estimated that the annual toxicity of metal emissions exceeds that of organics and radionuclides combined (Nriagu and Pacyna, 1988, Nature 333:134-138). Since soil and water contamination results in the uptake of heavy metals and toxins by crop plants, and eventually by humans, there is a pressing need for environmental cleanup to prevent entry of non-essential heavy metals into the food chain.
As sessile photosynthetic organisms, the mechanisms deployed by vascular plants for abrogating or alleviating heavy metal toxicity are of general interest. Not only does their lack of specialized excretory organs subject plants to large fluctuations in the levels of these substances and necessitate stringent intracellular homeostatic mechanisms, but the special status of plants as the principal points of entry of these substances into the food chain means that the mechanisms by which plants dispose of or sequester heavy metals have repercussions for all heterotrophic organisms.
In addition, bioremediation, the use of plants or microbes for the extraction and/or degradation of xenobiotics for environmental cleanup, is attracting increasing interest because of its potentially low cost by comparison with conventional physical and chemical methods. In the case of pollutants, such as heavy metals that cannot be degraded, phytoremediation is particularly appealing because of the ease with which plants can be harvested. Therefore, there is currently a great interest in the identification of native plant species or in the identification of genes from model systems useful for engineering crop species for increased resistance to and/or increased accumulation of heavy metals. In the latter category is the search for new heavy metal-binding peptides for the purpose of better understanding the mechanisms underlying the alleviation of heavy metal stress by plants and of obtaining genes encoding such peptides or the enzymes responsible for their synthesis. The identification and characterization of these genes will facilitate the development of a xe2x80x9cmix-and-matchxe2x80x9d approach to the manipulation of plant heavy metal responses according to the specific requirements of the type of environmental site that is to be phytoremediated.
To date, three classes of peptides have been shown to contribute to heavy metal resistance in plants: glutathione (GSH), metallothioneins (MTs), and phytochelatins (PCs). The thiol peptide, GSH (xcex3-Glu-Cys-Gly), and in some species its variant homoglutathione (h-GSH, xcex3-Glu-Cys-xcex2-Ala), is considered to influence the form and toxicity of heavy metals, such as As, Cd, Cu, Hg, and Zn, in several ways. These include the following mechanisms: direct metal binding (Fuhr and Rabenstein, 1973, J. Am. Chem. Soc. 95:6944-6950), promotion of the transfer of metals to other ligands (e.g, PCs and MTs; Freedman et al., 1989, J. Biol. Chem. 264:5598-5605), provision of reducing equivalents for the generation of metal oxidation states more amenable to binding by MTs and possibly PCs (Freedman et al., supra), removal of the active oxygen species formed as a result of exposure of cells to heavy metals (Inze and Van Montagu, 1995, Current Opinion in Biotech. 6:153-158), the formation of transport-active metal complexes with GSH (Li et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94:42-47), and by serving as a precursor for the biosynthesis of PCs and other cysteinyl peptides (Grill et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:439-443; Meuwly et al., 1995, Plant J. 7:391-400).
The MTs, another class of peptides previously implicated in heavy metal metabolism in plants, are small (4-8 kDa), cysteine-rich metal-binding polypeptides which are induced in cells by the presence of heavy metals. MTs, which contain multiple Cys-Xaa-Cys motifs, confer tolerance to a broad range of metals in mammals (Hamer, 1986, Annu. Rev. Biochem. 55:913-951) but appear to be involved primarily in Cu homeostasis in plants (Zhou and Goldsbrough, 1994, Plant Cell 6:875-884). Arabidopsis MT1 and MT2 confer tolerance to high levels of Cu2+ but only to low levels of Cd2+ when heterologously expressed in MT-deficient cup1 xcex94 mutants of S. cerevisiae (Zhou and Goldsbrough, 1994, Plant Cell 6:875-884). Further, MT expression in Arabidopsis seedlings is strongly induced by Cu2+ but not by Cd2+ (Zhou and Goldsbrough, 1994, Plant Cell 6:875-884; Murphy et al., 1997, Plant Physiol. 113:1291-1301), and comparisons between Arabidopsis ecotypes (i.e., subspecific forms in a true species, resulting from selection within a particular habitat, which can interbreed with other members of the species) demonstrate MT2 expression to be more closely correlated with Cu-tolerance than tolerance to other metals (Murphy and Taiz, 1995, Plant Physiol. 109:945-954).
Exposure of plants to heavy metals elicits the elaboration of PCs, a class peptides that play a pivotal role in heavy metal tolerance, primarily tolerance to Cd2+, in plants and fungi by chelating these substances and decreasing their free concentrations. PCs consist of repeating units of xcex3-glutamylcysteine followed by a C-terminal glycine {poly-(xcex3-Glu-Cys)n-Gly polymer} (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Steffens, 1990, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41: 553-575). Unlike MTs, PCs are synthesized posttranslationally from GSH (xcex3-Glu-Cys-Gly) by PC synthases (i.e., xcex3-glutamylcysteine dipeptidyl transpeptidases, EC 2.3.2.15), which transfer a xcex3-glutamylcysteine moiety from GSH to a second molecule or a previously synthesized PC molecule (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Zenk, 1996, Gene 179:21-30). Found in some fungi and in all plant species investigated to date (Rauser, 1990, Annu. Rev. Biochem. 59:61-86; Zenk, 1996, Gene 179:21-30), PCs bind heavy metals, such as Cd2+, with high affinity, and localize together with Cd2+ to the vacuole of intact cells (Vogeli-Lange and Wagner, 1990, Plant Physiol. 92:1086-1093). As indicated by the hypersensitivity of PC-deficient Arabidopsis cadl mutants to Cd2+ but not to Cu2+ (Howden et al., 1995, Plant Physiol. 107:1059-1066), PCs contribute most markedly to Cd2+ detoxification in planta. PC-dependent vacuolar Cd2+ sequestration is best understood in the fission yeast Schizosaccharomyces pombe, in which the hmt1+ gene product, a PC-selective ATP-binding cassette (ABC) transporter, pumps Cd.PCs and apo-PCs from the cytosol into the vacuole at the expense of ATP (Ortiz et al., 1992, EMBO J. 11:3491-3499; Ortiz et al., 1995, J. Biol. Chem. 270:47214728).
In addition to heavy metal tolerance mechanisms involving GSH, MTs, and PCs, the results of recent studies are consistent with the transport of free Cd2+ into the plant vacuole via a Cd2+/H+ antiport (Salt and Wagner, 1993,.J. Biol. Chem. 268:12297-12302). However, the physiological significance of such a process remains to be determined.
Although PCs play an important role in heavy metal tolerance in plants, the molecular identity of the enzyme(s) responsible for the elaboration of these peptides remained elusive. The prior art teaches the partial purification of heavy metal-, primarily Cd2+, activated enzymes (i.e., xcex3-glutamylcysteine dipeptidyl transpeptidases, EC 2.3.2.15, more commonly referred to as PC synthases) which are competent in the synthesis of PCs from GSH, homo-GSH or related thiol peptides by transfer of a xcex3-glutamylcysteine unit from one thiol tripeptide to another or to a previously synthesized PC molecule (Grill et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 89:6838-6842; Zenk, 1996, Gene 179:21-30). However, the prior art does not disclose the isolation or identification of the moiety or moieties involved in PC synthesis at either the protein or nucleic acid level.
Thus, despite the need for efficient and cost-effective methods for the bioremediation of contaminated soils and groundwaters, and the established importance of PCs for heavy metal accumulation and detoxification in plants, the identification and isolation of the enzyme(s) responsible for PC biosynthesis and for the phytoremediation of heavy metals has until now not been achieved. The present invention meets these need.
The invention includes an isolated nucleic acid encoding a phytochelatin synthase. In one aspect, the nucleic acid shares at least about 15% homology with at least one of AtPCS1 (SEQ ID NO: 1), AtPCS2 (SEQ ID NO: 3), TaPCS1 (SEQ ID NO: 5), SpPCS (SEQ ID NO: 7), and CePCS (SEQ ID NO: 9).
In another aspect, the isolated nucleic acid is selected from the group consisting of (SEQ ID NO: 1), (SEQ ID NO: 3), (SEQ ID NO: 5), (SEQ ID NO: 7), (SEQ ID NO: 9).
The invention further includes an isolated nucleic acid encoding a plant phytochelatin synthase, wherein the phytochelatin synthase shares at least about 15% homology with at least one of AtPCS1 (SEQ ID NO: 2), AtPCS2 (SEQ ID NO: 4), TaPCS1 (SEQ ID NO: 6), SpPCS (SEQ ID NO: 8), and CePCS (SEQ ID NO: 10).
The invention also includes an isolated polypeptide comprising a phytochelatin synthase. In one aspect, the isolated polypeptide shares at least about 15% homology with at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10.
In another aspect, the isolated polypeptide comprising a phytochelatin synthase is at least one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10.
In another aspect, the isolated nucleic acid encoding a phytochelatin synthase further comprises a reporter nucleic acid covalently linked thereto.
In yet another aspect, the reporter nucleic acid encodes a reporter polypeptide selected from the group consisting of a FLAG octapeptide, a human influenza virus hemagglutinin epitope, a xcex2-glucuronidase epitope, a green fluorescent protein epitope, and a luciferase epitope.
The invention includes a recombinant cell comprising an isolated nucleic acid wherein the nucleic acid shares at least about 15% homology with at least one of AtPCS1 (SEQ ID NO: 1), AtPCS2 (SEQ ID NO: 3), TaPCS1 (SEQ ID NO: 5), SpPCS (SEQ ID NO: 7), and CePCS (SEQ ID NO: 9).
In one aspect, the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
The invention also includes a vector comprising an isolated nucleic acid, wherein the nucleic acid shares at least about 15% homology with at least one of ATPCS1 (SEQ ID NO: 1), AtPCS2 (SEQ ID NO: 3), TAPCS1 (SEQ ID NO: 5), SpPCS (SEQ ID NO: 7), and CePCS (SEQ ID NO: 9).
The invention includes a transgenic plant, the cells, seeds and progeny of which comprise an isolated nucleic acid encoding a phytochelatin synthase, wherein the nucleic acid comprises at least one of AtPCS1 (SEQ ID NO: 1), AtPCS2 (SEQ ID NO: 3), TaPCS1 (SEQ ID NO: 5), SpPCS (SEQ ID NO: 7) and CePCS (SEQ ID NO: 9), or a fragment thereof, and wherein the nucleic acid shares at least about 15% homology with at least one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9.
The invention further includes a transgenic plant, the cells, seeds and progeny of which comprise an isolated nucleic acid encoding a phytochelatin synthase, or a fragment thereof, wherein the phytochelatin synthase shares at least about 15% homology with at least one of AtPCS1 (SEQ ID NO: 2), AtPCS2 (SEQ ID NO: 4), TaPCS1 (SEQ ID NO: 6), SpPCS (SEQ ID NO: 8), and CePCS (SEQ ID NO: 10).
The invention includes a method of preventing removal of a heavy metal from soil. The method comprises growing in the soil a transgenic plant comprising an isolated nucleic acid encoding a phytochelatin synthase in an antisense orientation and harvesting the plant from the soil, thereby preventing removal of the heavy metal from the soil.
The invention also includes a method of removing a heavy metal from soil. The method comprises growing in the soil a transgenic plant comprising an isolated nucleic acid encoding a phytochelatin synthase and harvesting the plant from the soil, thereby removing the heavy metal from the soil. In one aspect, the heavy metal is selected from the group consisting of cadmium, arsenate, arsenite, mercury, lead, zinc, nickel, bismuth, selenium, silver, gold, and copper.
The invention includes a method of generating a transgenic heavy metal resistant plant. The method comprises introducing to the cells of the plant an isolated nucleic acid encoding a phytochelatin synthase, thereby generating a transgenic heavy metal resistant plant.
The invention further includes a method of biosynthesizing a phytochelatin. The method comprises contacting an isolated phytochelatin synthase with a sufficient amount of glutathione, or a glutathione-related thiol peptide, under conditions which permit biosynthesis of a phytochelatin from the glutathione or related thiol peptide, thereby biosynthesizing a phytochelatin.
In one aspect, the biosynthesis is biosynthesis selected from the group consisting of a biosynthesis performed in vitro, and a biosynthesis performed in vivo, In another aspect, the glutathione-related thiol peptide is selected from the group consisting of a homoglutathione, a PC2, a PC3, a PC4, a homo-glutathione, a hydroxymethyl-glutathione, and a xcex3-glutamylcysteinylglutamic acid.
In yet another aspect, the phytochelatin is selected from the group consisting of a PC2, a PC3, a PC4, a homo-PC2, a hydroxymethyl-PC2, an iso-PC2(Glu), and a desGly-PC2.
In-even another aspect, the phytochelatin synthase is selected from the group consisting of a AtPCS1, a AtPCS2, a TaPCS1, a SpPCS, and a CePCS.
The invention includes a method of transferring a xcex3-glutamylcysteine unit from one thiol peptide to another. The method comprises contacting an isolated phytochelatin synthase with a glutathione, or a related thiol peptide, under conditions which permit transfer of the xcex3-glutamylcysteine unit from one thiol peptide to another, thereby transferring a xcex3-glutamylcysteine unit from one thiol peptide to another.
The invention also includes a method of decreasing the level of a heavy metal in a harvestable portion of a plant. The method comprises expressing a nucleic acid encoding a phytochelatin synthase in a non-harvestable portion of a plant, thereby decreasing the level of a heavy metal in the harvestable portion of the plant.
In one aspect, the method further comprises inhibiting expression of a phytochelatin synthase in the harvestable portion of the plant.
The invention includes a method of removing a heavy metal from groundwater. The method comprises growing in the groundwater a transgenic plant comprising an isolated nucleic acid encoding a phytochelatin synthase and harvesting the plant from the groundwater, thereby removing the heavy metal from the groundwater.